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Eun-Yi Oh,Mira Song,Mi-Hyun Ahn,Da-Young Park,Kyung-Jin Lee,Yangkang So,Zhe Lu,Kwang-Wok Min,Seul-Ki Lee,Jung-Hwan Lee,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Insect cell expression system using baculovirus has several benefits from high capacity, flexibility, and safety to humans and glycosylation capability. Thus, the baculovirus insect cell system has been widely used for production of recombinant protein. The HC and LC genes of the mAb CO17-1A were cloned under the control of two different promoters, P10 and PPH, respectively, on baculovirus expression pFastBacTM Dual vector. The gene expression cassettes carrying HC and LC genes were transferred into a parent bacmid in DH10Bac. Immunoflouorescence analyses and Western blot confirmed expression and secretion of mAb CO17-1A in the virus infected insect cells. The optimum condition for mAb expression was optimized at 24, 48 and 72hr after the virus infection with MOI ranging (0.2, 1 and 5). HPLC analysis revealed that the insect-derived mAb CO17-1A had insect specific glycan structures. Cell ELISA showed that the purified mAb from insect cell cultured media had a specific binding activity to SW948 cell. These results indicated that the baculovirus insect cell system is able to express, assemble, and secrete functional full size monoclonal antibody with insect specific glycosylation.
Da-Young Park,Yangkang So,Kyung-Jin Lee,Zhe Lu,Eun-Yi Oh,Kwang-Wok Min,Seul-Ki Lee,Jung-Hwan Lee,Mi-Hyun Ahn,Mira Song,Doo-Byoung Oh,Youngkwan Kim,Kisung Ko 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Plant genetic engineering has led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current antibody expression systems. In this study, the expression levels of mAbP SO57 with or without ER-signal peptides (Lys-Asp-Gly-Leu;KDEL) in transgenic tobacco plants were analysed in transgenic plant. PCR and Reverse Transcription-PCR analyses showed existence of heavy and light chain genes of mAb with or without KEDL and their transcription in plant, respectively. Western blot showed that the expression levels of mAbP SO57 with KDEL were significantly higher than that without KDEL. Flow cytometry analysis showed that the Fc domains of both purified mAbP and mammalian-derived mAb have similar binding activity to the FcγRI receptor. High-performance liquid chromatography analysis showed that the mAbP SO57 with KDEL had glycan profile with both oligomannose and golgi type, whereas the mAbP SO57 without KDEL had only golgi type glycans. Neutralizing analysis with rabies virus CVS-11 showed the similar neutralizing activity between mAbP SO57 with and without KDEL. These results suggest that the potential of mAbP SO57 for rabies immunotherapy is regardless of plant specific glycan structures.